Macroautophagic self-degradation (hereafter autophagy) is a common response of eukaryotic cells to stress stimuli like starvation or pathogen infection. Generally, bulk cytoplasm is non-selectively enclosed in autophagosomes, which are double membrane vesicles that fuse with lysosomes or the vacuole for degradation or recycling of the engulfed components. However, also specific targets can be degraded via receptors and adaptor proteins. During autophagosome formation, small ubiquitin-like proteins (UBLs) of the Atg8 family are covalently attached via their C-terminal Gly residue to phosphatidylethanolamine (PE) lipids on the autophagosomal membrane. Although it is clear that Atg8 lipidation and tethering to the autophagosomal membrane is essential for autophagosome biogenesis, the precise mechanism of Atg8 function so far remains elusive. Unlike S. cerevisiae that has only one Atg8 homolog, mammals encode two families of paralogous Atg8-like proteins (LC3 and GABARAP/GATE16) that may each contain several members and act as protein binding scaffolds in distinct steps of autophagosome formation. All Atg8 family members are structurally similar. Their structured core domain consists of an β-grasp fold preceded by two additional N-terminal α-helices and represents a versatile protein interaction surface that is essential for recruitment of the autophagy machinery to the autophagosomal membrane. The characteristic and flexible C-terminus ends with Phe-Gly (FG) or Tyr-Gly (YG). It is generated by Atg4 proteases that cleave C-terminally extended precursors. This group of highly specific proteases is also responsible for deconjugating Atg8 proteins from phosphatidylethanolamine (PE), a process that is required at a late stage of autophagosome formation.
As for Atg8, several paralogous Atg4-like proteases exist in higher eukaryotes, which might have different specificities for Atg8 paralogs (Li, M., Hou, Y., Wang, J., Chen, X., Shao, Z. M. and Yin, X. M. (2011) J Biol Chem 286, 7327-7338; Woo, J., Park, E. and Dinesh-Kumar, S. P. (2014) Proc Natl Acad Sci USA 111, 863-868). Amongst the four human Atg4 paralogs (Atg4A-D (Hemelaar, J., Lelyveld, V. S., Kessler, B. M. and Ploegh, H. L. (2003) J Biol Chem 278, 51841-51850; Kabeya, Y., Mizushima, N., Yamamoto, A., Oshitani-Okamoto, S., Ohsumi, Y. and Yoshimori, T. (2004) J Cell Sci 117, 2805-2812; Marino, G., Uria, J. A., Puente, X. S., Quesada, V., Bordallo, J. and Lopez-Otin, C. (2003) J Biol Chem 278, 3671-3678; Tanida, I., Sou, Y. S., Ezaki, J., Minematsu-Ikeguchi, N., Ueno, T. and Kominami, E. (2004) J Biol Chem 279, 36268-36276), Atg4B is the most versatile and active enzyme on recombinant fusion proteins. It can process the human Atg8 paralogs LC3B, GATE16, GABARAP and Atg8L with similar efficiencies (Li, M., Hou, Y., Wang, J., Chen, X., Shao, Z. M. and Yin, X. M. (2011) J Biol Chem 286, 7327-7338). The other three Atg4 enzymes are catalytically substantially less active. Solved structures of the free human Atg4B (Kumanomidou, T., Mizushima, T., Komatsu, M., Suzuki, A., Tanida, I., Sou, Y. S., Ueno, T., Kominami, E., Tanaka, K. and Yamane, T. (2006) J Mol Biol 355, 612-618; Sugawara, K., Suzuki, N. N., Fujioka, Y., Mizushima, N., Ohsumi, Y. and Inagaki, F. (2005) J Biol Chem 280, 40058-40065) and LC3B-bound Atg4B (Satoo, K., Noda, N. N., Kumeta, H., Fujioka, Y., Mizushima, N., Ohsumi, Y. and Inagaki, F. (2009) EMBO J 28, 1341-1350) show that the protease has a papain-like fold with an additional unique domain participating in the protease's interaction with the folded substrate domain. The flexible C-terminus of Atg8-like substrates makes additional contacts to a pocket on the protease surface that directs the substrates' C-terminal Gly residues into active site. The protease's flexible N-terminus may fold back onto the substrate-binding pocket and has therefore been suggested to negatively regulate substrate interaction (Satoo, K., Noda, N. N., Kumeta, H., Fujioka, Y., Mizushima, N., Ohsumi, Y. and Inagaki, F. (2009) EMBO J 28, 1341-1350). The human Atg4B protease also contains a flexible extension at the C-terminus. In substrate-free structures (Kumanomidou, T., Mizushima, T., Komatsu, M., Suzuki, A., Tanida, I., Sou, Y. S., Ueno, T., Kominami, E., Tanaka, K. and Yamane, T. (2006) J Mol Biol 355, 612-618; Sugawara, K., Suzuki, N. N., Fujioka, Y., Mizushima, N., Ohsumi, Y. and Inagaki, F. (2005) J Biol Chem 280, 40058-40065), this extension is poorly resolved and folds back on the substrate interaction surface, which might suggest that it interferes with substrate binding. To obtain crystals of substrate-bound Atg4B, the C-terminal extension had to be deleted (Satoo, K., Noda, N. N., Kumeta, H., Fujioka, Y., Mizushima, N., Ohsumi, Y. and Inagaki, F. (2009) EMBO J 28, 1341-1350). Its functional relevance so far remained elusive. Atg8-like proteins represent only one class of UBLs. This larger group of small protein modifiers also includes the founding member ubiquitin, SUMO and NEDD8 that act as regulators of various intracellular processes (reviewed in van der Veen, A. G. and Ploegh, H. L. (2012) Annu Rev Biochem 81, 323-357; and Yeh, E. T., Gong, L. and Kamitani, T. (2000) Gene 248, 1-14). In contrast to Atg8-like proteins, other UBLs, however, generally possess a C-terminal Gly-Gly (GG) motif and are conjugated to proteins by isopeptide bonds formed between their C-terminal carboxyl group primary amine groups on the surface of target proteins. Importantly, all mentioned UBLs are initially processed and often deconjugated by dedicated proteases (van der Veen, A. G. and Ploegh, H. L. (2012) Annu Rev Biochem 81, 323-357). In most cases, these proteases are highly efficient, which can be exploited for biochemical applications. The yeast SUMO specific protease Ulp1, for example, has successfully been used for the in vitro tag-removal from recombinant proteins (Malakhov, M. P., Mattern, M. R., Malakhova, O. A., Drinker, M., Weeks, S. D. and Butt, T. R. (2004) J Struct Funct Genomics 5, 75-86). Recently, the inventors characterized additional UBL-specific proteases and found that the Brachypodium distachyon (bd) SUMO- and NEDD8-specific proteases bdSENP1 and bdNEDP1 remove tags even more robustly and with an up to 1000 times higher efficiency than TEV protease (Frey, S. and Görlich, D. (2014) J Chromatogr A 1337, 95-105; Frey, S. and Görlich, D. (2014) J Chromatogr A 1337, 106-115). Importantly, bdSENP1 and bdNEDP1 display mutually exclusive (i.e. orthogonal) substrate specificity and can thus be used for the highly efficient purification of recombinant proteins and stoichiometric protein complexes by on-column or post-column cleavage (Frey, S. and Görlich, D. (2014) J Chromatogr A 1337, 95-105; Frey, S. and Görlich, D. (2014) J Chromatogr A 1337, 106-115). The application of UBL-specific proteases in eukaryotic systems is typically hampered by cross-reactivity with endogenous UBL-processing enzymes. Recently, the SUMO variant SUMOstar has been introduced, which allows purification of recombinant fusion proteins also from eukaryotic hosts (Liu, L., Spurrier, J., Butt, T. R. and Strickler, J. E. (2008) Protein Expr Purif 62, 21-28; Peroutka, R. J., Elshourbagy, N., Piech, T. and Butt, T. R. (2008) Protein Sci 17, 1586-1595). Further UBL substrates that are stable in eukaryotic hosts might become valuable tools that can be used for the purification of protein complexes (Frey, S. and Görlich, D. (2014) J Chromatogr A 1337, 106-115). In other applications, such substrates may be used for the induced in-vivo cleavage of recombinant fusion upon intracellular expression of the respective protease. Such in-vivo manipulation can e.g. be applied to modify the stability or localization of a protein of interest (Taxis, C. and Knop, M. (2012) Methods Mol Biol 832, 611-626; Urabe, M., Kume, A., Takahashi, T., Serizawa, N., Tobita, K. and Ozawa, K. (1999) Biochem Biophys Res Commun 266, 92-96; Taxis, C., Stier, G., Spadaccini, R. and Knop, M. (2009) Mol Syst Biol 5, 267). Tag-removing proteases are powerful tools in protein biochemistry. Although several proteases are routinely used for this purpose (Malakhov, M. P., Mattern, M. R., Malakhova, O. A., Drinker, M., Weeks, S. D. and Butt, T. R. (2004) J Struct Funct Genomics 5, 75-86; Butt, T. R., Edavettal, S. C., Hall, J. P. and Mattern, M. R. (2005) Protein Expr Purif 43, 1-9; Arnau, J., Lauritzen, C., Petersen, G. E. and Pedersen, J. (2006) Protein Expr Purif 48, 1-13; Li, S. J. and Hochstrasser, M. (1999) Nature 398, 246-251; Nilsson, J., Stahl, S., Lundeberg, J., Uhlen, M. and Nygren, P. A. (1997) Protein Expr Purif 11, 1-16; Young, C. L., Britton, Z. T. and Robinson, A. S. (2012) Biotechnol J 7, 620-634), most of them have severe drawbacks including low specific activity, limited specificity or strict constraints concerning temperature, buffer requirements or sequence context. Recent work from the inventors has introduced bdSENP1 and bdNEDP1, two new proteases that are largely devoid of these limitations (Frey, S. and Görlich, D. (2014) J Chromatogr A 1337, 95-105). The inventors recently also described the application of the S. cerevisiae (sc) Atg4 protease for tag removal (Frey, S. and Görlich, D. (2014) J Chromatogr A 1337, 95-105). scAtg4 is highly active in vitro and displays mutually exclusive cleavage specificity to SUMO, NEDD8 and ubiquitin-processing enzymes. Unfortunately, however, neither this protease nor scAtg8 fusion proteins are well behaved in terms of solubility and/or expression level.
WO 2002/090495, WO 2003/057174, WO 2005/003313, and WO 2006/073976 disclose the use of SUMO and other UBLs for increasing expression levels of proteins. WO 2005/003313 and WO 2008/083271 further mention that UBLs can be cleaved using SUMO proteases.
The amino acid sequence of xlAtg4B is known from UniProt sequence Q640G7. It is an object of the present invention to provide new proteases that could potentially be used for tag removal. More specifically, the inventors were interested to find well-behaved and stable protease fragments with optimal proteolytic activity.